This competitive renewal proposes to continue our work on the regulation of mammalian protein-encoding genes with a focus on post-transcriptional processes and, in particular, nonsense-mediated mRNA decay (NMD). NMD generally occurs when translation terminates sufficiently upstream of an exon-junction complex (EJC) during a pioneer round of translation. We define the pioneer translation initiation complex as bound by the cap-binding protein heterodimer CBP80-CBP20 and, if the mRNA derived from pre-mRNA splicing, one or more EJCs, and we have shown that both CBP80 and EJCs promote NMD. Over the last ~4 years we have continued to characterize changes that typify the pioneer translation initiation complex during the process of NMD. For example, we have shown that the key NMD factor, the ATP-dependent RNA helicase called UPF1, binds to most if not all cellular transcripts at a low level nonspecifically. However, a series of carefully choreographed steps leads to the activation of NMD target-bound UPF1 by phosphorylation. We have found that phosphorylated UPF1 binds specifically to NMD-target 3'-untranslated regions (3'UTRs) and, unlike steady-state UPF1, which is largely hypophosphorylated, provides the first reliable identifier of natural cellular NMD targets. We have also demonstrated that cellular DNA damage by commonly used chemotherapeutics triggers the caspase-mediated cleavage of UPF1 so as to generate a dominant-negative C-terminal cleavage product; this product then downregulates the efficiency of NMD so as to upregulate a battery of natural NMD targets that we have found using transcriptome deep-sequencing include those encoding apoptotic-promoting proteins. In fact, cells can be primed to undergo faster and more efficient apoptosis upon exposure to an NMD inhibitor followed by its withdrawal prior to chemotherapy. By working with the Singer lab during her last year with me, Hana Sato used new Singer-lab technologies that track individual transcripts in intact cells to show that the NMD of premature termination codon (PTC)-containing -globin mRNA occurs on the cytoplasmic-side of the nuclear envelop and not in the nucleoplasm or after release into the cytoplasm. In other studies, we have collaborated to examine in cultured cells and in rats how human UPF1 expression overcomes cytotoxicity or paralysis, respectively, caused by proteins implicated in amyotrophic lateral sclerosis. In this proposal, AIM 1 is to conduct a genome-wide inventory to define new NMD factors using haploid genetics. This AIM will supplement our on-going studies of the mechanism of NMD. AIM 2 is to identify new roles for NMD factors based on our unpublished data. We aim to investigate putative roles for (i) UPF1 together with Tudor-SN in microRNA decay, (ii) CBP80 in the PGC1-mediated upregulation of estrogen receptor- and orphan estrogen- related receptor-activated genes, and (iii) UPF1 together with FMRP, FRX1 and FXR2 on mRNA metabolism in fragile X mental retardation syndrome. AIM 3 is to develop therapeutics for the many and diverse dominantly inherited diseases that are due to PTCs that fail to trigger NMD and generate toxic truncated proteins.